Polysaccharides are high molecular weight carbohydrates, composed by one or more monosaccharides that form repeating units and polymerize. They are the most abundant macromolecules among living organisms, being present in all plants and algae, in several animals and in some microorganisms. Due to their physical-chemical properties, namely, their water retention capacity, film forming and rheology (viscosity, gelling, emulsifying, etc.), polysaccharides are largely used in a wide variety of industrial applications.
Currently, polysaccharides obtained from plants (ex. Guar gum, Arabic gum, pectins), algae (ex. alginates, carrageenan, agar) or crustaceous (ex. chitin) dominate the biopolymers market, where microbial polysaccharides still represent a small fraction. The main factors limiting microbial polysaccharide wider use are associated with their production costs, mainly substrate cost, and also to the fact that many of the producing strains are pathogenic or it is difficult to obtain public acceptance for some applications. Nevertheless, during the last years, there has been an increasing interest in isolating and identifying new microbial polysaccharides that may compete with traditional polysaccharides due to their physical-chemical properties and rheology. The production of plant and algal polysaccharides, in particular, is subjected to climatic and environmental impact, such as pollution, that cause great variability both in the quantity and the quality of the polymers obtained. On the other hand, many microbial polysaccharides are characterized by a variety of properties not found in plant polymers, such as, for example, anti-tumor, antiviral, anti-inflammatory or immune-stimulating activities.
Microbial polysaccharides that have been extensively studied and are currently being commercially exploited include: bacterial cellulose, produced by Acetobacter xylinum, whose properties are similar to plant cellulose; dextran, produced by bacteria of the Leuconostoc genus, and levan, produced by bacteria of the genera Bacillus, Zymomonas and Lactobacillus, which are exclusively bacterial products; xanthan, produced by bacteria of the Xanthomonas genus, and gellan gum, produced by Sphingomonas paucimobilis, which have improved physical properties compared to traditional polysaccharides, such as alginate or carrageenan; hyaluronic acid, produced by Streptococcus equii, and succinoglycan, produced by bacteria of the genera Pseudomonas, Rhizobium, Agrobacterium and Alcaligenes that find medical, pharmaceutical and cosmetic applications due to their similarity to eukaryotic polymers.
Due to the growing interest in renewable resources as alternatives to chemical products, the search for new products will certainly be intensified and new microbial polysaccharides with commercial interest are likely to arise. The commercial value of a polysaccharide will depend on its composition, on the amount produced and the ease of extraction and processing. The industrial development will depend on its rheological properties, namely its ability to form viscous solutions, stability for wide temperature and pH ranges, and on its unique biological properties and/or the fact that they may be used in new applications.
Galactose-rich polymers may be included among the polysaccharides with potential industrial interest. These polymers may be found in plants (ex. Arabic gum), algae (ex. carrageenan and agar) and in several microorganisms, including protozoa, fungi, yeast and bacteria. The presence of galactose residues in microbial polymers is rather common, even though the type of glycosyl linkage involved varies. These polysaccharides may be homopolymers of galactose (galactans) or heteropolymers containing, besides variable amounts of galactose, other sugar residues, most commonly glucose, mannose, rhamnose, arabinose and/or fucose. Many of these polymers contain, besides neutral sugars, acidic sugars (ex. glucuronic acid, galacturonic acid) or amino-sugars (ex. N-acetyl-glucosamine, N-acetyl-galactosamine). The presence of non-sugar components, such as acyl groups (ex. acetate esters, pyruvate ketals, succinyl half esters) or inorganic residues (ex. sulphate, phosphate) is also quite common.
Galactose homopolymers are produced by bacteria such as Bifidobacterium infantis (Tone-Shimokawa et al., 1996), Bifidobacterium catenulatum (Nagaoka at al., 1996), Klebsiella pneumoniae (Whitfield et al., 1991), Pasteurella haemolytica (Lacroix et al., 1993), Serratia marcescens (Aucken at al., 1998), Azorhizobium caulinodans (D'Haeze et al., 2004) and Methylobacterium sp. VTT-E-11929 (Verhoef at al., 2003).
The main component of the polymer of the invention is a heteropolysaccharide, containing besides galactose as its main component, other neutral sugars, namely, glucose, mannose and rhamnose, which confer it a higher structural complexity. Unlike the galactans produced by Bifidobacterium infantis and Bifidobacterium catenulatum, wherein the galactose residues are present in the form of furanose rings, the polysaccharide of this invention contains all galactose residues in the form of pyranose rings. On the other hand, the extraction process of the referred galactans is rather difficult since they are cell wall components, whereas the extraction of the polymer of the invention is much easier because it is an extracellular product. The galactans produced by Klebsiella pneumoniae, Pasteurella haemolytica and Serratia marcescens are lipopolysaccharides, composed by alternate pyranose and furanose rings of galactose. These bacteria are pathogenic for Man (K. pneumoniae and S. marcescens) and animals (P. haemolytica), being the galactans produced by them associated with their infection development.
For this reason, the interest in these polymers is restricted study of the pathogenesis of the infections caused by the producing bacteria, being their commercial development unlikely. Besides, taken that they are lipopolysaccharides, the extraction and purification process is more difficult than for the extracellular polymer of the invention.
Heteropolymers containing galactose as their main component are produced by a wide group of microorganisms, namely, bacteria of the genera Bifidobacterium, Klebsiella, Erwinia, Methylobacterium, Pseudomonas, Lactobacillus, Alcaligenes and Streptococcus. 
Rhamnogalactans (polysaccharides composed by galactose and rhamnose) are common cell wall components of bacteria of the Bifidobacterium genus. An example of this is the cell wall polysaccharide of Bifidobacterium longum that is composed by galactose (about 60%) and rhamnose (about 40%), both in the form of pyranose rings (Nagaoka at al., 1995). The polymer of the invention, besides its extracellular nature, differs from the Bifidobacterium longum polymer by having a lower percentage of rhamnose and also by having other neutral sugars (glucose and mannose).
Some bacteria of the Klebsiella genus produce galactose-rich extracellular heteropolymers such as: Klebsiella sp. strain K32 that produces a polysaccharide composed of galactose (45-63%) and rhamnose (12-55%), with a variable pyruvate content (Bryan et al., 1986); Klebsiella sp. S11 that produces a polysaccharide composed of galactose (62.5%), glucose (25%) and mannose (12.5%), with a minor content of uronic acids (Dermlim et al., 1999); and Klebsiella planticola DSM 3092 that produces a polysaccharide composed of galactose (38.2%), mannose (15.9%), glucose (1.7%), glucuronic acid (17.5%), acetate (5.3%), succinate (2.6%) and sulphate (14.6%) (EP0184755). The polymer of the invention differs from these polysaccharides by its composition, namely, the simultaneous presence of galactose, glucose, mannose and rhamnose, and the absence of uronic acids, which distinguishes it from the polysaccharide produced by Klebsiella planticola. 
The production of galactose-rich heteropolymers also occurs in bacteria of the Methylobacterium genus. An example is the extracellular polysaccharide methylan, produced by Methylobacterium organophilum, composed by galactose, glucose and mannose (in the molar ratio 4:3:3), acyl groups (pyruvate and acetate) and uronic acids (U.S. Pat. No. 5,064,759). The polymer of the invention differs from methylan polysaccharide by its higher galactose content and by the absence of uronic acids.
Among the phytopathogenic bacteria of the Erwinia genus, some produce galactose-rich polysaccharides. Examples thereof include: Erwinia amylovora produces amylovoran, an extracellular polysaccharide composed by galactose (about 80%) and glucuronic acid (about 20%), acyl groups (acetate and pyruvate) and traces of glucose (Eastgate at al., 2000); Erwinia pyrifoliae produces an extracellular polysaccharide, similar to amylovoran, but with a higher acetate content and without glucose (Kim et al., 2002); Erwinia stewartii (Pantoea stewartii sap. stewartii) produces stewartan, a capsular polysaccharide similar to amylovoran but with a higher glucose content (Minogue et al., 2005); Erwinia chrysanthemi Ech6 produces an extracellular polysaccharide composed by galactose and fucose, in equal amounts, glucose and glucuronic acid (Yang et al., 2001). The polymer of the invention differs from these polymers by the fact that it does not contain glucuronic acid and, also, by its mannose and rhamnose contents.
Several Enterobacter species (ex. Enterobacter amnigenus, Enterobacter cloacae) produce heteropolysaccharides rich in galactose (21-24%) and fucose (16-27%), containing variable amounts of glucose, mannose and rhamnose, acyl groups (acetate and pyruvate) and uronic acids (glucuronic acid and/or galacturonic acid) (Verhoef et al., 2005). Colanic acid, which is composed by galactose, fucose, glucose and glucuronic acid, is a typical extracellular polysaccharide produced by bacteria of the Enterobacter genus (Ratto et al., 2006). The polymer of the invention differs from these polysaccharides by its higher galactose content, trace or null fucose content and the absence of uronic acids.
The production of galactose-rich heteropolysaccharides also occurs in bacteria of the Vibrio genus, such as, for example, Vibrio harveyi, that produces a polysaccharide whose main components are galactose and glucose, with minor amounts of rhamnose, fucose, ribose, arabinose, xylose and mannose (Bramhachari at al., 2006). This polysaccharide also has a high content of uronic acids, namely, galacturonic acid that distinguishes it from the polymer of the invention.
Bacteria from the Alcaligenes genus, namely the strain Alcaligenes l ATCC 31961, were referred as having the ability of producing a polysaccharide containing typically glucose and rhamnose, but also glucuronic, galactose, mannose, arabinose, fucose and ribose (EP0471597A1). The polymer of the invention differs from that, since it does not contain uronic acids.
Several lactic acid bacteria from the genera Lactobacillus, Lactococcus and Streptococcus produce a wide variety of heteropolysaccharides whose main components are galactose and glucose. These species include: Lactobacillus delbrueckii that produces several polysaccharides containing, besides galactose and glucose, rhamnose or mannose; Lactobacillus rhamnosus and Lactobacillus kefiranofaciens that produce polysaccharides containing galactose and glucose; Lactobacillus paracasei that produces a polysaccharide containing galactose and N-acetyl-galactosamine (Faber at al., 2001; Vanhaverbeke et al., 1998; Yang, 2000); Lactococcus lactis ssp. cremoris that produces polysaccharides composed by galactose and glucose or composed by galactose, glucose and rhamnose (Yang, 2000); Streptococcus species that produce several polysaccharides containing galactose and glucose, rhamnose, mannose or N-acetyl-galactosamine (Yang, 2000); Streptococcus thermophilus produces polysaccharides containing galactose and rhamnose (Vaninggelgem et al, 2004) or polysaccharides containing galactose, rhamnose and glucose (U.S. Pat. No. 5,965,127).
The production of galactose-rich polysaccharides also occurs in bacteria of the Pseudomonas genus, such as, for example: Pseudomonas marginalis that produces marginalan, an extracellular polysaccharide composed by galactose and glucose in equal molar amounts (Osman at al., 1989); Pseudomonas fluorescens that produces an extracellular polysaccharide whose main components are galactose, mannose and arabinose (Hung at al., 2005); Pseudomonas paucimobilis that produces a polysaccharide containing typically glucose and rhamnose, but also glucuronic, galactose, mannose, arabinose, fucose and ribose (EP0471597); and Pseudomonas species ATCC 53923 that produces a polysaccharide containing mannose galactose and glucose in a molar ratio of 1.3:1.0:1.3, 10-2 uronic acids and 10-15% acetate (EP0410604). The polymer of the invention differs from marginalan because it contains, besides galactose and glucose, also mannose and rhamnose, as main components. The presence of arabinose in the polysaccharide produced by Pseudomonas fluorescens distinguishes it from the polysaccharide of the invention, in which arabinose is absent or is present in trace amounts. The polymer of the invention also differs from those produced by Pseudomonas paucimobilis and Pseudomonas species ATCC 53923 mainly because they do not contain uronic acids.
The polymer of the invention has a composition that distinguishes it from other galactose-rich polysaccharides from microbial origin, namely, because it has, besides galactose as the main component, the neutral sugars glucose, mannose and rhamnose, further lacking uronic acids and amino-sugars.
The polymer of the invention is an extracellular product, which makes its extraction a relatively easy process, comparing to some of the polysaccharides that are constituents of the bacterial cell-wall or the plant or algae cell-walls.
Due to its biodegradability, the galactose-rich polymer does not cause any environmental problems. The polysaccharide of the invention has interesting rheological properties, namely, its behavior as a pseudoplastic fluid and its ability to form aqueous solutions with excellent viscosity, stable for wide pH and temperature ranges.
Although, both the composition and the amount of polysaccharide produced by a microorganism are genetically determined traits, it is possible to influence both by altering the culture conditions. Polysaccharide production may be induced as part of a stress response, being generally favored by: presence of carbon source in excess, concomitant with limitation by another nutrient (ex. nitrogen or phosphorus); low temperatures; microaerophilic or anaerobic conditions or excessive aeration; saline stress; presence of cations (ex. Ca2+ or Sr2+); or the presence of toxic compounds or microbial growth inhibitors (ex. antibiotics or H2O2). The amount of polysaccharide produced is influenced by the medium composition and the incubation conditions, especially, the carbon availability, both intra and extracellular, and the ratio between carbon and other nutrients.
Most fermentation processes for the production of extracellular microbial polysaccharides are performed with pure cultures (ex. EP0410604, ES8701838, US2004/0197877). Nevertheless, it is possible to use mixed cultures of two or more microorganisms of which at least one is able to produce the polymer of interest. An example of this is the production of extracellular polysaccharides by a mixed culture of Pseudomonas maltophilia DSM 2130 and Agrobacterium tumefaciens DSM 2128 (U.S. Pat. No. 4,567,140).
Microbial polysaccharide production is usually performed by aerobic fermentation, being sugars (ex. glucose, sucrose, starch) the most commonly used carbon sources. Most processes described above for microbial galactose-rich polysaccharides used sugars as carbon sources, mainly glucose, or, in some cases, sucrose or lactose. For methylan production, by Methylobacterium organophilum, methanol was used as carbon source, or alternatively, mixtures containing methanol and glucose, mannose, galactose or succinate. The process of the present invention uses glycerol or glycerol-rich substrates as carbon source for the microbial fermentation. The use of glycerol is advantageous since it allows for the valorization of glycerol wastes (ex. glycerol-rich product from the biodiesel production), thus reducing the production costs associated with carbon source. The process of the invention also considers the use of other carbon sources (ex. sugars, methanol) as alternatives to glycerol or mixture thereof, which makes the process much more versatile.
In an aerobic fermentation, in which the culture broth viscosity continuously increases, reaching a highly viscous state, one of the main difficulties of the process is maintaining an efficient distribution of oxygen and nutrients across the broth. This is, frequently, achieved by keeping high aeration rates and/or high stirring rates. On the other hand, viscosity reduction to enhance mass transfer and polymer recovery may be achieved by adding nucleases to cell lysates or using engineered microbial strains that produce those enzymes. In fact, bacteria such as Ralstonia eutropha, Methylobacterium organophilum, Aeromonas caviae, Azotobacter vinelandii, Alcaligenes latus, Escherichia coli and Klebsiella, as well as some from Pseudomonas genus, have been genetically manipulated to produce nucleases during the production of polyhydroxyalkanoates and polysaccharides (WO 99/50389). The process differs from that of this invention in the type of polysaccharide produced and the carbon source used. In the process of this invention, the production of the galactose-rich polymer is performed with low dissolved oxygen concentrations, allowing for the minimization of aeration and, subsequently, reduction of operation costs.
The co-production of extracellular polysaccharides and intracellular biopolymers, namely, polyhydroxyalkanoates (PHA), occurs naturally in several microorganisms, under specific growth conditions. Examples of microorganisms capable of simultaneously producing polysaccharides and PHA, include: bacteria of the Rhizobium genus (ex. Rhizobium meliloti), that accumulate intracellular reserves of polyhydroxybutyrate (PHB), and produce an extracellular polysaccharide composed by glucose, galactose and glucuronic acid (Tavernier et al., 1997); the bacteria Azotobacter vinelandii and Pseudomonas aeruginosa that produce an extracellular polysaccharide, alginate, and accumulate intracellular PHB (Galindo et al., 2007; Pham et al., 2004). The process of the present invention may be used for the production of intracellular biopolymers, namely PHA, simultaneously with the production of the galactose-rich extracellular polymer.
The recovery of extracellular microbial polysaccharides usually involves the separation of the cells, following the precipitation of the polymer by the addition of a solvent miscible with water in which the polymer is insoluble (ex. EP0410604). Depending on the intended use for the polymer, it may be further subjected to additional processes for purification. On the other hand, there are some applications for which there is no need for a high degree of purity and the polymer may be used directly from the culture broth (ex. US2006/0147582).
Polysaccharides are used in a large range of applications, such as in medicine and food, pharmaceutical and chemical industries (U.S. Pat. No. 0,197,877A1).
In food industry, galactose-rich polysaccharides may be applied as thickening, binding, gelling, texturing, emulsifying and stabilizing agents in liquid Systems, such as salad dressings, vinegar, ice-cream, ketchup, mustard, dehydrated products (ex. soups, sauces, cereals and pap meals) and meat-based products (ex. sausages). In the pharmaceutical industry, they have been used as binding agents and for drug controlled release.
Some microbial polysaccharides present flocculating activity, and may be used alone or mixed with other biopolymers, such as chitin derivates, galactomannans, glucomannans, alginates and starches (EP0471597A1). Flocculating agents are useful in colloid and cell aggregation, being currently used in industrial applications, such as water treatment and food and mining industries. Inorganic and synthetic organic flocculating agents are inexpensive products, but have a low biodegradability. On the other hand, some of them are dangerous for human health, namely polyacrilamides, whose monomers are neurotoxic, and poly(aluminium chloride) that induces Alzheimer disease. Although natural flocculating agents usually have a lower flocculating activity, they are safe and biodegradable, and its application will certainly increase in the near future.
A large percentage of the polymeric compounds produced by microorganisms, like polysaccharides, have the capacity of immobilizing toxic metals. This ability depends on the chemical composition and molecular structure of the biopolymer. Bacterial polysaccharides, such as alginate and xanthan gum, are able to immobilize actinides (ex. plutonium) forming erosion resistant aggregates. The use of microbial polysaccharides for toxic metal removal from contaminated soil and water has a great potential, and the interest in its application has been increasing.
The galactose-rich polysaccharides, namely Guar gum, are currently used in other areas, such as paper industry, for paper properties enhancement (paper strength and surface improvement for printing); explosives, as binding agent in blasting slurries and water proofing agent in stick explosives (ex. ammonium nitrate, nitro-glycerine); petroleum industry, as suspending agent in well drilling; hydromulching, incorporated in the tackifier portion of the slurry used; and textile industry, as thickener for die.
Due to their biodegradability, polysaccharides have also found application in the preparation of films for packaging. Biopolymers, such as alginate, chitosan, starch, gellan and pectin, have been used in the development of biodegradable films for food packaging, since they present a low permeability to gases (carbon dioxide and oxygen).
The galactose-rich polysaccharides can also be converted into oligosaccharides (polymers that contain from 2 to 10 monomers) that may be used in the food industry. The interest in using these natural compounds as prebiotics (non-carcinogenic, non-digestible and low caloric compounds that stimulate the development of benefit microflora in the digestive tract) has been increasing, as traditional food additives are becoming less popular among consumers. Nowadays, the best strategy to obtain oligosaccharides in large quantities is based on the degradation of polysaccharides using physical treatments (microwave, heating, radiation, sonication), chemical treatments (acid hydrolysis), enzymatic reactions (using microbial enzymes) or by the action of specific microorganisms.
General Description of the Invention
The present invention concerns a biopolymer, whose main component is a high molecular weight polysaccharide, composed by galactose, glucose, mannose and rhamnose. The polysaccharide may, additionally, contain xylose, fucose, ribose, arabinose and/or fructose, in trace amounts, and non-sugar components, such as acyl groups. The polymer of the invention is insoluble in organic solvents and forms highly viscous aqueous solutions, with pseudoplastic fluid behavior. The viscosity of the polymer in aqueous solution is stable for a pH range of 3-10, decreasing as the temperature increases from 0° C. to 100° C. The polymer of the invention has both flocculating and emulsifying activities, and film-forming capacity.
The present invention also describes a process for the production of the galactose-rich polymer, by microbial fermentation, using glycerol or glycerol-rich substrates as carbon sources, in an aerated and stirred bioreactor. The process according to the invention also foresees the use of other carbon sources (ex. sugars, alcohols, organic acids or alkanes), as alternatives to glycerol or in mixtures with glycerol. The process of the invention further foresees the use of food or industrial waste, such as, for example, glycerol-rich product from biodiesel production, whey or olive-oil production wastes.
The microbial culture used in the fermentation process of this invention is a bacterium, for example, from the genera Pseudomonas, Klebsiella, Methylobacterium, Erwinia, Alcaligenes, Lactobacillus, Streptococcus or Ralstonia. The microbial culture is preferably a Pseudomonas oleovorans strain. The microbial culture is a wild type microorganism, a variant or a mutant, as long as it is able to produce the galactose-rich polymer. It is possible to use a pure culture or a mixed culture of several microorganisms, among which, at least one is capable of producing the galactose-rich polymer.
The fermentation process used for the production of the galactose-rich polymer consists of growing a microbial culture in an aerated aqueous nutrient medium. The fermentation starts with a high dissolved oxygen concentration, but, concomitantly with cellular growth, it is gradually reduced, being controlled below 30%, preferably, below 10% or null. The galactose-rich polymer is produced under conditions of nitrogen limitation and carbon availability, simultaneously with the maintenance of a low dissolved oxygen concentration.
The present invention describes the recovery of the galactose-rich polymer at the end of the fermentation, by the direct use of the culture broth, after a drying process. The present invention also describes the process for the extraction of the galactose-rich polymer in its native form, just as well as its purification process. The process of extraction of the polymer of this invention consists of cell removal by centrifugation of the culture broth, following the precipitation of the polymer by addition of a precipitating agent (ex. ethanol, acetone). The purification of the polymer involves the use of one or more additional processes (ex. dialysis, ultrafiltration or diafiltration of aqueous solution of the polymer).
This invention also concerns the use of the galactose-rich polymer in several food and industrial applications (ex. pharmaceutical, mining, paper, textile, explosive, etc.) and its use as a source of oligosaccharides and in the preparation of biodegradable films.